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DTs-ll is a highly diphtheria toxin (DT)-sensitive cell line previously isolated by transfection of wildtype DT-resistant mouse L-M(TK-) cells with the cDNA encoding a monkey Vero cell DT receptor. DTs-II (Mr 37,195). The cytotoxic action ofDT occurs by the following steps: (i) binding to specific cell-surface receptors, (ii) internalization of the (toxin-receptor) complexes into vesicles, and (iii) translocation of the A fragment from acidified vesicles into the cytosol, where it inhibits protein synthesis by ADP-ribosylation of elongation factor 2 (1-3).Our laboratory has recently used expression cloning to identify a receptor for DT (4). The gene encoding the receptor was cloned by transfecting wild-type toxin-resistant mouse L-M(TK-) cells with a cDNA library prepared from highly toxin-sensitive monkey Vero cells; the transfectants were screened for DT sensitivity employing a replica plate system that allowed for the detection of those mouse cells whose protein synthesis is inhibited upon exposure to DT and that, at the same time, preserved a "replica" ofthose cells (4,5

Genetic and biochemical evidence that sedoheptulose-7-phosphate is an obligatory precursor of the L-glycerc-D-mannoheptose residues of the lipopolysaccharide of Salmonella was obtained by isolation and characterization of transketolase-negative mutants of Salmonella typhimurium. These mutants, which are defective in synthesis of sedoheptulose-7-phosphate, were found to produce an incomplete heptose-deficient lipopolysaccharide, and were also sensitive to bile salts, a characteristic property of heptose-deficient mutants. Phenotypic repair of the defect in lipopolysaccharide synthesis was obtained by addition of exogenous sedoheptulose-7-phosphate to growing cultures of the mutant strains. Characterization of revertants isolated either as transketolase-positive or heptose-positive provided further evidence that the heptose deficiency resulted from mutation at the transketolase locus. On the basis of these findings a possible pathway for conversion of sedoheptulose-7-phosphate to L-glycero-Dxmannoheptose is proposed.L-Glycero-D-mannoheptose is a characteristic component of the lipopolysaccharides of SalmoneUa and related Gramnegative bacteria, and is a specific and major constituent of the innermost backbone region of rthe polysaccharide (Fig. 1). The pathway of biosynthesis of this sugar is unknown, although it has been assumed to be derived from sedoheptulose-7-phosphate (1, 2). To test this hypothesis we needed a mutant incapable of making sedoheptulose-7-P.While several reactions leading to formation of sedoheptulose-7-P are known, all depend on the presence of transketolase (TK): TK ribose-5-P + xylulose-5-P it sedoheptulose-7-P + glyceraldehyde-3-P [1] TK ribose-5-P + fructose-6-P 2::± sedoheptulose-7-P + erythrose-4-P TK fructose-6-P + glyceraldehyde-3-P >::xylulose-5-P + erythrose-4-P TA erythrose-4-P + fructose-6-P T± sedoheptulose-7-P + glyceraldehyde-3-P Ald erythrose4-P + dihydroxyacetone-P 2± sedoheptulose-1,7-diP [5]SDPase or sedoheptulose-1,7-diPsedoheptulose-7-P + Pi [6] FDPase In addition to reactions 1 and 2, catalyzed by transketolase, sedoheptulose-7-P can be derived from erythrose-4-P by reactions involving either transaldolase (reaction 4) or aldolase (reactions 5 plus 6); however, the only known sources of erythrose-4-P for these alternate pathways are reactions 2 and 3, which are themselves catalyzed by transketolase.Transketolase-negative mutants should therefore be deficient in synthesis of sedoheptulose-7-P, and, if this sugar is an obligatory precursor of L-glycero-D-mannoheptose, should produce an incomplete, heptose-deficient lipopolysaccharide. We now report that this is indeed the case, and that the defect in lipopolysaccharide synthesis can be corrected in vivo by addition of exogenous sedoheptulose-7-P to the culture medium.Isolation of transketolase-negative mutants was first described by Josephson and Fraenkel (3) in Escherichia coli. These workers developed a method of selection based on the fact that transketolase is essential both for catabolism of pentoses and for sy...

Monkey (Mk) CD9 antigen has been shown previously to increase the diphtheria toxin (DT) sensitivity of cells when co-expressed with Mk proHB-EGF (DT receptor). We have elucidated here the mechanism whereby Mk CD9 influences Mk proHB-EGF and present evidence that Mk CD9 is a coreceptor for DT. We observed that Mk CD9 not only increased the DT sensitivity but also increased the DT receptor affinity of cells. Furthermore, the higher the Mk CD9/Mk proHB-EGF ratio, the higher the affinity. In contrast, mouse (Ms) CD9 did not increase the toxin sensitivity or receptor affinity of cells when co-expressed with Mk proHB-EGF. Using Mk/Ms chimeric CD9 molecules, we determined that the second extracellular domain of Mk CD9 is responsible for both increased sensitivity and receptor affinity. This domain of Mk CD9 also interacts with Mk proHB-EGF in a yeast two-hybrid system. Our findings thus suggest that Mk CD9 has a direct physical interaction with Mk proHB-EGF to form a DT receptor complex and that this contact may change the conformation of the receptor to increase DT binding affinity and consequently increase toxin sensitivity. We thus propose that Mk CD9 is a coreceptor for DT.

SummaryThe transmembrane precursor of the monkey (Mk) heparin-binding, epidermal growth factor-like growth factor (proHB-EGF) functions as a diphtheria toxin (DT) receptor, whereas the mouse (Ms) precursor does not. Previously, using chimeric Ms/Mk precursors, we have shown that DT resistance of cells bearing Ms proHB-EGF may be accounted for by several amino acid substitutions between residues 122 and 148 within the EGF-like domain and that Glu-141 is an important amino acid residue for DT binding. In this study, reciprocal site-directed mutagenesis was performed on the major non-conserved residues in the region of 122-148, alone or in combination, between Mk and Ms precursors to identify more precisely which amino acid residues are important for DT binding. Two approaches were used. The first, more traditional approach was to destroy DT sensitivity and binding of Mk proHB-EGF by substitution(s) with the corresponding Ms residue(s). From the single mutations, the greatest loss of DT sensitivity was observed with Mk/Glu-141His (approximately 4000-fold) and the next greatest with Mk/Ile-133Lys (approximately fourfold). The double mutations Mk/Leu-127Phe/Glu-141His, Mk/Ile-133Lys/ Glu-141His and Mk/His-135Leu/Glu-141His resulted in complete toxin resistance (> 100 000-fold). The second approach, both novel and complementary, was to gain DT binding and sensitivity of Ms proHB-EGF by substitution(s) with the corresponding Mk residue(s). Surprisingly, the single mutation Ms/His-141Glu resulted in the gain of moderate DT sensitivity (> 260-fold). The double mutation Ms/Lys-133Ile/His-141Glu and the triple mutation Ms/Lys-133Ile/Leu-135His/His-141Glu resulted in a progressive gain in toxin sensitivity (> 4700-fold and > 16 000-fold respectively) and affinity. This triple mutant cell line is essentially as sensitive (IC 50 ¼ 3.1 ng ml ¹1 ) as the highly toxin-sensitive monkey Vero cell line (IC 50 ¼ 4 ng ml ¹1 ), indicating that these three Mk residues enable the Ms proHB-EGF to act as a fully functional DT receptor. Taken together, these results indicate that Glu-141 plays the most critical role in DT binding and sensitivity and that two additional amino acid residues, Ile-133 and His-135, also play significant roles.

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